Phytochemical profile , antimicrobial potential and GC-MS analysis of wild variety of Olea Europaea ( Olive ) cultivated in Pakistan

Olive plant produces a variety of bioactive molecules and thus has important medicinal value in folk medicine. In this study, different leaf and fruit extracts of Pakistani wild variety of Olea europaea was tested for their phytochemical content, antimicrobial activity and mass spectrometric analysis. Olive leaves and fruit samples were extracted with five different solvents to obtain the crude extract and screened for various kinds of phytochemicals. Phytochemicals were further confirmed through Fourier Transmission Infra-red Spectroscopy (FTIR). The plant extract showed significant antimicrobial activity against all the strains tested. Methanol, ethanol and ethyl acetate extracts were found more effective against most of the pathogenic bacteria with high zone of inhibition. Gas Chromatography-Mass Spectrometry (GC-MS) analysis revealed that olive fruits have Oleic acid, Palmitic acid, Linoleic acid, Octadecadienoic acid, Stearic acid, Palmitoleic acid and Tridecanoic acid as oil contents. In this work, the antimicrobial potential and phytochemical contents were explored which may further pave the way for the bio-industrial applications.


Introduction
Plants have been used as medicines since the dawn of civilization for thousands of years [1].About 80% of population in developing countries uses traditional medicine for curing different diseases.So, the investigation of these plants is helpful in understanding their characteristics, usefulness and effective nature [2].Chemical constituents of plants have been exploited for the discovery of therapeutic agents as well as new sources of such economical materials as tannins, oils, gums, forerunners for the production of complex chemical substances.The olive tree is amongst the oldest known cultivated trees in the world that has been an important source of nutrition and medicine [3].Olive is a broad-leaved, evergreen tree of the family Oleaceae that is present in native coastal areas of the Mediterranean region.The olive has been used generally in customary medications in European Mediterranean islands and countries such as Spain, Israel, Morocco, France and Greece where it is cultivated mainly as edible oil and table olives [4].Bacteria are serious pathogens and cause a wide variety of human diseases including cholera, leprosy, bacterial pneumonia, whooping cough, and diphtheria.Bacterial pathogens are also a serious threat to the food industry [5].Antibiotics provide main basis for the therapy of microbial infections.However, the emergence and spreading of bacterial resistance has made the treatment of infectious diseases more problematic [6].
The antimicrobial activity of plants is highly related to secondary substances that are synthesized and produced by plants The aim of the study was to investigate the antimicrobial activities and phytochemical contents utilizing different methods in order to compare with the reported literature.The data produced here may be used in production of biomolecules of medical importance.

Collection of plant materials
The fresh leaves of olive plant were collected from district Dir of Khyber Pakhtunkhwa, Pakistan.The rinsed olive leaves of collected samples were air dried under shade and then converted to fine powder by crushing in electronic grinder.The fine powdered form of the plants was then kept in airtight glass containers to be protected from different contaminants until used for further analysis and screening.

Preparation of leaves extracts
The olive leaves were air dried and ground into powder form and then 100 (g) of the powder was extracted with 350 mL of ethyl acetate, ethanol, methanol, distilled water and hexane each (Technical grade-Merck) and boiled water in 1000 mL conical flasks.Flasks were vigorously shaken at 400 rpm overnight in a Labotec model 20.2 shaking machine.After shaking, the supernatant was decanted into pre-weighed, labeled flasks.The process was repeated three times to exhaustively extract the leaves materials.

Identification of phytochemicals by GC-MS analysis
About 100 g olive seed material was extracted through soxhlet extraction apparatus with 250 ml hexane.The extract was concentrated by removing solvent under reduced pressure in a rotary evaporator.According to AOAC standard reference method, derivatization was performed prior to GC-MS analysis [16].About 25 mg fat was mixed with 0.1 ml internal standard (1.37 mg) and 1.5 ml of sodium hydroxide solution in methanol (0.5 N), preserved and warmed in boiling water bath for 5 min.The hydrolyzed sample was chilled and mixed with 2.5 ml of boron triflouride solution in methanol (10%).The solution was then conserved and warmed in boiling water bath for 30 min and chilled.The esterified solution was mixed with 5 ml saturated sodium chloride solution and extracted two times with 1 ml n-hexane.The filtration of n-hexane extract is done with 0.45 µm membrane filter and injected 1 µl to GC-MS through auto injector system.GC-MS analysis was performed on GC-MS Model QP 2010 plus (Tokyo, Japan) equipped with an auto-sampler (AOC-20S) and auto-injector (AOC-20i  Further confirmation about phytochemicals was done by spectra obtained using Fourier Transmission Infra-Red Spectroscopy (FTIR) model (IRPrestige-21, Shimadzu Corporation Kyoto Japan) (Figure 1) which revealed that polar phytochemicals were separated in high amount with polar solvents and non-polar constituents with non-polar solvents.
In this spectra, the phenols, tannins, carbohydrates and proteins were absent in hexane extract due to non-polar nature of hexan.While methanol and ethanol extracts had highest amount of flavonoides and terpenoids due to the polar nature of the solvent which is according the like dissolve like concept of solvents.The antimicrobial activities revealed that ethyl acetate and methanol extracts of olive exhibited maximum activity while minimum antimicrobial activities were observed for hexane extracts (Figure 3).It is because of the fact that alcohol and ethyl acetate extracts give rise to flavonoids and phenolic phytochemicals as compared to hexane which were confirmed from the Fourier transmission Infra-red Spectroscopy (FTIR) spectra (Figure 1).The results from the disc diffusion method, followed by measurement of ZOI, indicated that the methanol extract showed strong inhibitory activity against Bacillus Atrophus, with the highest inhibition zones (10±0.40mm)which is in the range with its antibiotic counterpart.(16±0.21mm)(Figure 3).The inhibitory activity of these extracts confirmed the antimicrobial activity and its potential use in the treatment of microbial diseases.The GC-MS chromatogram of Olive fruit oil contents revealed the presence of saturated and unsaturated fatty acids as shown in Figure 4.The fragmentation patterns obtained from GC-MS analyzer was compared with that of National Institute of Standard and Technology (NIST) library through which 13 different fatty acids were identified.The different fatty acids identified based on GC-MS are given in Table 3. Oleic acid was found in highest concentration (30.00%), while the other fatty acids were; Palmitic acid (11.65%),Linoleic acid (6.50%),Octadecadienoic acid (5.98%),Stearic acid (5.30%),Palmitoleic acid (1.66%) and Tridecanoic acid (1.38%).
The rest of the fatty acids were present in less than 1% concentration (Table 3).Similar chromatographic analysis has been done for olive plant of different countries and various biomolecules were evaluated for their biopharmaceutical application [7].The olive tree naturally possesses strong antimicrobial activity which has been utilized in traditional medicine to fight fever and overcome infections[8].It is desirable to get knowledge about the bioactive constituents of plants like fatty acids because of their nutritional value, diagnosis of definite diseases and pharmacology.Various analytical techniques like Spectrophotometry, HPLC and gas chromatography (GC) have been applied for the analysis of fatty acids [9-12].GC-MS is a useful method for the determination of fatty acids due to its high speed, resolution and sensitivities [13].

Figure 1 .Figure 2 .
Figure 1.FTIR spectra of olive plant leaves for phytochemicals 25, 26].GC-MS analysis revealed that olive oils contain different biologically active compounds like fatty acids thus olive plant, besides its antibacterial and antifungal activities can also be used to produce different pharmaceutical products to cure diseases including cancer.

Figure 3 .Figure 4 .
Figure 3. Antimicrobial activity of olive leaves extracts along with standard antibiotics